Characterization of the stress degradation products of tolvaptan by UPLC-Q-TOF-MS/MS

Article abstract of DOI:10.1039/c4ra16644b

Tolvaptan (TVT) is a selective, competitive vasopressin receptor 2 antagonist used to treat hyponatremia. TVT was subjected to forced degradation under hydrolysis, oxidation, dry heat and photolysis conditions, in accordance with the ICH guideline Q1A (R2). The degradation products (DPs) formed have been characterized through UPLC-PDA and UPLC-Q-TOF-MS/MS studies. The chromatographic separation was achieved on an Acquity UPLC HSS T3 column (100 × 2.1 mm, 1.7 μm) with a mobile phase containing a gradient mixture of solvents A (0.1% formic acid) and B (acetonitrile) at a flow rate of 0.3 ml min^-1 at 30 °C. The detection wavelength was set at 266 nm. The drug degraded under acid hydrolysis, base hydrolysis and oxidative conditions to form a total of 7 DPs. When methanol was used as the co-solvent during stress degradation, four additional DPs were formed which were absent when acetonitrile was used as the co-solvent. Comparison of the fragmentation pattern of the DPs with that of the drug helped in the elucidation of the structures of all the degradation products. The degradation pathway of the drug was established, which was duly justified by the mechanistic explanation. The developed UPLC method was validated as per ICH guidelines.

Full text of DOI:10.1039/c4ra16644b

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Characterization of the stress degradation products
of tolvaptan by UPLC-Q-TOF-MS/MS†
Cite this: RSC Adv., 2015, 5, 21142
a
b
a
Prinesh N. Patel, D. Rajesh Kumar, S. Gananadhamu and R. Srinivasab
*
Tolvaptan (TVT) is a selective, competitive vasopressin receptor 2 antagonist used to treat hyponatremia.
TVT was subjected to forced degradation under hydrolysis, oxidation, dry heat and photolysis conditions,
in accordance with the ICH guideline Q1A (R2). The degradation products (DPs) formed have been
characterized through UPLC-PDA and UPLC-Q-TOF-MS/MS studies. The chromatographic separation
was achieved on an Acquity UPLC HSS T3 column (100 ꢀ 2.1 mm, 1.7 mm) with a mobile phase
containing a gradient mixture of solvents A (0.1% formic acid) and B (acetonitrile) at a flow rate of 0.3
ml minꢁ1 at 30 ꢂC. The detection wavelength was set at 266 nm. The drug degraded under acid
hydrolysis, base hydrolysis and oxidative conditions to form a total of 7 DPs. When methanol was used as
the co-solvent during stress degradation, four additional DPs were formed which were absent when
acetonitrile was used as the co-solvent. Comparison of the fragmentation pattern of the DPs with that of
the drug helped in the elucidation of the structures of all the degradation products. The degradation
pathway of the drug was established, which was duly justified by the mechanistic explanation. The
developed UPLC method was validated as per ICH guidelines.
Received 18th December 2014
Accepted 5th February 2015
DOI: 10.1039/c4ra16644b
www.rsc.org/advances
Analytical methods for estimation of TVT include esti-
1. Introduction
mation in biological samples6–10 and in formulation.11
A
Tolvaptan (TVT) is chemically known as (ꢃ)-40-[(7-chloro-2,3,4,5-
tetrahydro-5-hydroxy-1H-1-benzazepin-1-yl)carbonyl]-o-tolu-m-
toluidide, and is an oral non-peptidic selective vasopressin V2-
receptor antagonist.1–4 It is indicated for the treatment of clin-
ically relevant hypervolemic or euvolemic hyponatremia asso-
ciated with heart failure, cirrhosis, or syndrome of
inappropriate antidiuresis.1,2 TVT selectively inhibits arginine
vasopressin (AVP) induced water reabsorption in the kidney by
competitively blocking the binding of AVP to V2-receptors in the
distal nephron, thereby preventing the antidiuresis caused by
circulating AVP.3–5
The characterization of degradation products is an impor-
tant task in the drug discovery and development process. The
international conference on harmonization (ICH) guidelines
emphasizes the identication of the likely degradation prod-
ucts, degradation pathways and intrinsic stability of a molecule.
Hence, it is necessary to carry out stability studies of a drug
under various forced degradation conditions of hydrolysis,
oxidation, thermal and photolysis.
liquid chromatography-tandem mass spectrometry method
for determining tolvaptan and its nine metabolites in rat
serum has also been reported, which involves gradient
elution using 0.3% acetic acid and acetonitrile with 0.3%
acetic acid on a C18 column employing ESI positive ioniza-
tion mode.12 Stability methods by UPLC13 and HPLC14 for the
impurity proling of TVT are reported, by which the identi-
cation of degradation products have not been attempted.
Govind Reddy et al. have reported a RP-HPLC method for the
estimation of process impurities in the presence of degra-
dation products with only three known DPs, whereas others
were not characterized.15 In the present work, to better
understand the mechanism of degradation of TVT and
characterize the degradation products, a chromatographic
method involving a diode array detector (DAD) and tandem
mass spectrometry (MS/MS) is developed. The degradation of
TVT according to the ICH guidelines has been performed and
the mechanism for the TVT degradation has been
established.
aDepartment of Pharmaceutical Analysis, National Institute of Pharmaceutical
Education and Research (NIPER), Balanagar, Hyderabad, 500037, Telangana, India.
2. Experimental
E-mail: gana@niperhyd.ac.in; Fax: +91-4023073751; Tel: +91-4023073740; +91- 2.1. Chemicals and reagents
4023073741
Pure tolvaptan was supplied by MSN Laboratories limited
bNational Centre for Mass Spectrometry, CSIR-Indian Institute of Chemical
(Hyderabad, India). Commercially available 15 mg tolvaptan
tablets (TOLVAT, Sun Pharmaceuticals, Mumbai, India) were
purchased from a local pharmacy. HPLC grade acetonitrile
Technology, Hyderabad, 500 007, Telangana, India
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra16644b
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(ACN), methanol (MeOH) and formic acid were purchased
For LC-MS characterization, an Agilent 1290 series LC
from Merck (Mumbai, India). Ammonium acetate (HPLC instrument (Agilent Technologies, USA) attached to a quadru-
buffer grade) and hydrogen peroxide (30% w/v) were pole-time of ight (Q-TOF) mass spectrometer (Q-TOF-LC/MS,
purchased from Finar Chemicals Pvt Ltd. (Ahmedabad). 6540 series, Agilent Technologies, USA) equipped with an elec-
Analytical reagent (AR) grade sodium hydroxide (NaOH) and trospray ionization (ESI) source was used. Data analysis was
hydrochloric acid (HCl) were purchased from Fine Chemicals carried out using Mass Hunter workstation soware. The typical
Limited, Ahmedabad. High purity water was prepared using a operating source conditions were optimized as follows: the
Millipore Milli-Q Plus water purication system (Millipore, fragmentor voltage was set at 140 V, the capillary at 3500 V, and
Milford, MA, USA).
the skimmer at 65 V; nitrogen was used as the drying (325 ꢂC; 10
l minꢁ1) and nebulizing (40 psi) gas. All the spectra were
recorded under identical experimental conditions, and are an
average of 25 scans. MS scans were carried out in positive ESI
mode.
A photo stability chamber (Osworld), consisting of both UV
and uorescent lamps, was used for the photo degradation
study. A water bath equipped with a temperature controller was
used to carry out degradation studies for all the solutions. A
2.2. Instrumentation
The UPLC system used was an Acquity UPLC H Class system
from Waters (Waters, Milford, MA, USA) with a quaternary
solvent manager, autosampler, column oven and PDA detector.
Data acquisition, analysis, and reporting were performed using
Empower3 (Waters) chromatography soware.
Fig. 1 Overlay of chromatograms showing effect of co-solvent: [A] acid hydrolysis, [B] base hydrolysis, [C] oxidative degradation.
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ꢂ
controlled temperature dry air oven (Osworld laboratory oven) and auto sampler temperatures were maintained at 30 C and
ꢂ
was used for the solid-state thermal stress study.
5 C, respectively. An injection volume of 1 ml was used.
2.3. Chromatographic conditions
2.4. Preparation of stock and standard solutions
The chromatographic separations were carried out on an Acq- A standard stock solution of TVT (500 mg mlꢁ1) was prepared in
uity UPLC HSS T3 column (100 ꢀ 2.1 mm, 1.7 mm) with a mobile HPLC grade ACN. Aliquots of the stock solution were trans-
phase containing a gradient mixture of solvents A (0.1% formic ferred and diluted with ACN : water (50 : 50 v/v) to yield
acid) and B (acetonitrile) at a ow rate of 0.3 ml minꢁ1. The concentrations equal to 25, 50, 75, 100, 125 and 150 mg mlꢁ1
.
gradient programme was set as follows (time in min/% For the assay of the tablets, 20 tablets were crushed. Aer
proportion of solvent B): 0–0.5/25, 0.50–2.50/45, 2.50–3.50/55, mixing, an amount equivalent to one tablet was weighed and
3.50–5.50/60, 5.50–6.50/70, 6.50–7.00/25, 7.00–9.00/25. The taken in a 100 ml volumetric ask, dissolved in diluent
eluted compounds were monitored at 266 nm. The column oven (ACN : water (50 : 50% v/v)), sonicated for 15 min, diluted up to
Scheme 1 Proposed structures of protonated degradation products of tolvaptan.
Table 1 MS/TOF data of tolvaptan and degradation products along with their elemental composition and major fragments
Retention time
(min)
Molecular formula
Observed m/z
Calculated m/z
Error
MS/MS fragment ions
+
TVT
6.55
3.18
4.08
4.40
4.98
5.48
5.53
6.22
6.24
6.93
7.41
7.45
C
26H27ClN2O3
449.1620
212.0833
331.1206
345.1365
270.1121
431.1535
445.2140
435.1485
313.1096
284.1273
447.1481
180.0585
449.1626
212.0837
331.1208
345.1364
270.1125
431.1521
445.2122
435.1470
313.1102
284.1281
447.1470
180.0575
1.34
1.89
0.60
431, 252, 206, 180, 119, 91, 65
180, 164, 152, 144, 130, 117, 103
313, 206, 180, 134
238, 206, 180, 134, 106
226, 178, 119, 91, 65
339, 252, 206, 178, 119
427, 252, 202, 176, 119
417, 238, 180, 105
DP-1
DP-2
DP-3
DP-4
DP-5
DP-6
DP-7
DP-8
DP-9
DP-10
DP-11
C11H14ClNO+
C18H20ClN2O2
+
+
C19H22ClN2O2
ꢁ0.29
+
C16H16NO3
1.48
+
+
C26H24ClN2O2
ꢁ3.25
ꢁ4.04
ꢁ3.45
1.92
+
C27H29N2O4
C25H24ClN2O3
C
18H17ClN2O+
206, 178, 134, 106
252, 192, 164, 136, 119, 105
252, 222, 119, 91, 65
+
C17H18NO3
2.82
+
C
C
26H24ClN2O2
ꢁ2.46
10H11ClN+
ꢁ5.55
164, 152, 144, 130, 117, 103
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the mark with the same solvent and ltered through a 0.45 mm
Whatman lter paper. From this solution, further dilutions
were made with diluent to obtain a solution containing
100 mg mlꢁ1. This solution was analyzed for the assay.
2.5.2. Oxidative degradation. TVT was dissolved in 1 ml of
ACN and treated with a solution of 10% (v/v) H2O2 at room
temperature in the dark for 24 h.
2.5.3. Photolytic degradation. For the solid sample,
approximately 100 mg of TVT was spread on a petri-dish in a
layer of 1 mm thickness. For the solution, the drug was initially
dissolved in ACN, and then diluted with water. The samples
were kept in a photo-stability chamber and exposed to
1.2 million lux h of uorescent light and 200 Wh mꢁ2 of UV
illumination as per ICH conditions8 for 3 days. A parallel set of
the drug samples were stored in the dark at the same temper-
ature to serve as a control.
2.5. Forced degradation studies
2.5.1. Hydrolysis. For acid hydrolysis, 5 mg drug was dis-
solved in 1 ml of ACN, then 4 ml of 1 N HCl was added and the
solution was maintained at reux at 80 ꢂC for 24 h. The studies in
alkaline conditions were carried out by dissolving 5 mg of drug in
1 ml of ACN, followed by the addition of 4 ml of 2 N NaOH, and
then the solution was reuxed at 80 ꢂC for 17 h. For neutral
hydrolysis, the drug was initially dissolved in ACN, then diluted
with water and the solution was reuxed at 80 ꢂC for 48 h.
Aer completion of the degradation treatments, the samples
were allowed to cool to room temperature, neutralized as needed
2.5.4. Thermal degradation. The dry heat degradation was
carried out by exposing the drug to a temperature of 80 ꢂC for 7
days in a hot air oven.
2.6. Effect of co-solvent on degradation
and injected into the chromatographic system aer dilution with Initially, ACN was used as the co-solvent for all stress degrada-
the diluent (ACN : water – 50 : 50, v/v) to 250 mg mlꢁ1
tion studies. As MeOH is a cheaper solvent, it was also tried as a
.
Fig. 2 ESI/MS/MS spectra of (a) TVT (m/z 449) at 10 eV, (b) DP-1 (m/z 212) at 10 eV, and (c) DP-2 (m/z 331) at 10 eV.
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co-solvent for sample preparation for stress degradation methanol, which is clear from the chemical structures of the
studies.
aforementioned degradation products.
3. Results and discussion
3.1. Method development
3.4. UPLC-MS/MS of TVT and its degradation products
3.4.1. MS/MS of TVT. To elucidate the degradation
behavior of TVT (retention time (Rt) ¼ 6.55 min), the ESI-MS/MS
spectrum (Fig. 2a) of its [M + H]+ ion (m/z 449) was examined.
The spectrum shows abundant product ions at m/z 431 (loss of
H2O), m/z 252 (loss of C10H12ClNO), m/z 206 (loss of C15H15NO
from m/z 431), m/z 180 (loss of CO from m/z 206), m/z 119 ((2-
methylbenzylidene)oxonium), and m/z 91 (loss of CO from m/z
119) (Scheme 2). It can be noted that the product ions at m/z 206
and 180 are characteristic of the 7-chloro-5-hydroxy-2,3,4,5-tet-
rahydro-1H-benzo[b]azepine-1-carbonyl skeleton of TVT, while
the fragment ions with m/z 252 and 119 are diagnostic of the 3-
methylphenyl-2-methylbenzamide skeleton of TVT. The
elemental compositions of all these ions have been conrmed
by accurate mass measurements.
3.4.2. MS/MS of degradation products. On-line LC-ESI-MS/
MS experiments were performed to characterize all the degra-
dation products (DP-1 to DP-11) formed under the various stress
conditions. The most probable structures have been proposed
for all the degradation products based on the m/z values of their
[M + H]+ ions and the MS/MS data in combination with the
In order to achieve the optimum resolution, numerous changes
to the chromatographic conditions, such as the pH and
composition of the mobile phase, ow rate, column, etc., were
carried out. Different mobile phase compositions using buffers
at pH 3.0, pH 4.0, and pH 5.5, and 0.1% formic acid with ACN
and MeOH were tried. Isocratic methods were not successful in
achieving a favorable resolution. Well resolved degradation
product peaks, along with a sharp and symmetrical drug peak,
were obtained using 0.1% formic acid in water and ACN, using
the proposed gradient method with a UPLC HSS T3 column. For
LC/MS analysis, electrospray ionization (ESI) and atmospheric
chemical ionization were tried in both positive and negative
modes. It was observed that the maximum intensity was
obtained with ESI in positive mode. Mass spectrometer condi-
tions were optimized to obtain maximum ionization of TVT and
all the DPs. Experiments were conducted using different capil-
lary voltages (3000 V, 3500 V and 4000 V) and different frag-
mentor voltages (100 V, 125 V, 140 V, 170 V) at a constant source
ꢂ
temperature of 325 C. The maximum intensity of the analytes
was observed when using a capillary voltage and a fragmentor
voltage of 3500 V and 140 V, respectively.
3.2. Degradation behavior of TVT
The optimized LC-MS method was used to identify the degra-
dation products of TVT. The overlay of the chromatograms of
the stress degradation samples are given in Fig. 1. A total of
eleven DPs were identied and characterized using LC/ESI/MS/
MS experiments and accurate mass measurements. The
proposed structures of the DPs and their elemental composi-
tions are given in Scheme 1 and Table 1.
3.2.1. Hydrolysis. The drug degraded signicantly to form
DP-1, DP-2, DP-3, DP-4, DP-8, DP-9 and DP-11 under acid
hydrolysis. Under alkaline hydrolysis conditions, TVT degraded
to yield DP-1, DP-2, DP-4 and DP-5, whereas it was stable even
aer reuxing under neutral hydrolytic conditions for 48 h.
3.2.2. Oxidative degradation. The drug was degraded to
form minor degradation products DP-6, DP-7, DP-10.
3.2.3. Photolytic degradation. No degradation was
observed under photo-degradation conditions.
3.2.4. Thermal degradation. The exposure of the solid drug
to a temperature of 80 ꢂC for 7 days did not result in signicant
decomposition, which indicates that TVT is stable to dry heat.
3.3. Effect of co-solvent on degradation
As shown in the Fig. 1, DP-1, DP-3 & DP-9 under acid hydrolysis,
DP-1 under base hydrolysis and DP-6 under oxidative degrada-
tion were formed when MeOH was used as the co-solvent.
However, they were not formed when exposed to the same stress
conditions using ACN as the co-solvent. This suggests that DP-1,
DP-3, DP-6 and DP-9 were formed due to the presence of Scheme 2 Proposed fragmentation pathway of protonated tolvaptan.
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elemental compositions derived from accurate mass measure- 134 (loss of C10H10ClN). Based on all these data, the 4-amino-(2-
ments, as discussed below. methylphenyl)(7-chloro-5-hydroxy-2,3,4,5-tetrahydro-1H-benzo[b]-
DP-1. The ESI-MS/MS spectrum of the [M + H]+ ion (m/z 212) azepin-1-yl)methanone structure can be proposed for DP-2.
of DP-1 (Rt ¼ 3.18 min) is given in Fig. 2b. The MS/MS spectrum
DP-3. The ESI-MS/MS spectrum of the [M + H]+ ion (m/z 345)
shows structure indicative fragment ions as shown in Scheme of DP-3 (Rt ¼ 4.40 min) is shown in Fig. 3a. The spectrum
3(a). The peak at m/z 180 is diagnostic for 7-chloro-2,3-dihydro- displays product ions at m/z 238 (loss of C7H9N), m/z 206 (loss of
1H-benzo[b]azepine, which further fragments to give m/z 144 CH3OH from m/z 238, ((7-chloro-2,3-dihydro-1H-benzo[b]aze-
(loss of HCl). As shown in Scheme 4(a), the mass difference of 32 pin-1-yl)-methylidyne)-oxonium), which are compatible with the
between the [M + H]+ ion of DP-1 (m/z 212) and DP-11 (m/z 180) structure (4-amino-2-methylphenyl)(7-chloro-4-methoxy-2,3,4,5-
indicates the addition of methanol to DP-11. Based on all these tetrahydro-1H-benzo[b]azepin-1-yl)methanone (Scheme 3(b)).The
data, DP-1 was identied as 7-chloro-4-methoxy-2,3,4,5-tetra- mechanism of formation of DP-3 is shown in Scheme 4(a). It can
hydro-1H-benzo[b]azepine.
be noted that the mechanism may involve an addition of
DP-2. The ESI-MS/MS spectrum of the [M + H]+ ion (m/z 331) methanol to DP-8.
of DP-2 (Rt ¼ 4.08 min) (Fig. 2c) with an elemental composition
DP-4. The ESI-MS/MS spectrum (Fig. 3b) of the [M + H]+ ion of
of C18H20ClN2O2 shows product ions which are given in Scheme DP-4 (Rt
¼
4.98 min.) with an elemental composition
C16H16NO3 displays product ions which are compatible with
+
3(b). The characteristic product ions of DP-2 include m/z 313
(loss of water), m/z 206 (loss of C7H9N from m/z 313), and m/z the structure 2-methyl-4-(2-methylbenzamido)benzoic acid. The
Scheme 3 Proposed fragmentation pathway of protonated tolvaptan degradation products: (a) DP-1 and DP-11; (b) DP-2, DP-3, DP-7 and DP-8;
(c) DP-5, DP-6 and DP-10; (d) DP-4 and DP-9.
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Scheme 4 Probable mechanisms of formation: (a) DP-1–DP-5, DP-8, DP-9 and DP-11;(b) DP-6, DP-7 and DP-10.
Fig. 3 ESI/MS/MS spectra of (a) DP-3 (m/z 345) at 15 eV, (b) DP-4 (m/z 270) at 15 eV and (c) DP-5 (m/z 431) at 20 eV.
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Table 2 High resolution mass spectrometry (HRMS) data of product Table 2 (Contd.)
ions of protonated tolvaptan and its degradation products
Molecular formula Observed m/z Calculated m/z Error
Molecular formula Observed m/z Calculated m/z Error
+
DP-4
C16H16NO3
270.1121
226.1217
178.0490
431.1535
445.2140
427.2018
202.0860
176.1080
435.1485
417.1360
238.0887
105.0358
313.1096
178.0416
284.1273
192.0643
136.0759
447.1481
222.0311
180.0585
164.0269
144.0818
130.0662
270.1125
226.1226
178.0499
431.1521
445.2122
427.2016
202.0863
176.1070
435.1470
417.1364
238.0863
105.0335
313.1102
178.0418
284.1281
192.0655
136.0757
447.1470
222.0316
180.0575
164.0262
144.0808
130.0651
1.48
3.98
+
15H16NO+
TVT
C
C
C
26H27ClN2O3
449.1611
431.1512
252.1006
206.0356
180.0567
119.0494
91.0540
212.0833
180.0574
164.0266
152.0249
144.0795
130.0649
117.0563
331.1206
313.1090
180.0572
134.0594
106.0653
345.1365
238.0608
449.1626
431.1521
252.1004
206.0367
180.0575
119.0491
91.0542
212.0837
180.0575
164.0262
152.0262
144.0808
130.0651
117.0573
331.1208
313.1102
180.0575
134.0600
106.0651
345.1364
238.0629
3.34
2.09
C
+
+
26H25ClN2O2
C9H8NO3
5.05
+
+
16H14NO2
ꢁ0.79
DP-5
DP-6
C26H24ClN2O2
ꢁ3.25
ꢁ4.04
ꢁ0.47
1.48
C11H9ClNO+
C10H11ClN+
C8H7O+
5.34
+
C27H29N2O4
+
4.44
C27H27N2O3
+
ꢁ2.52
C
12H12NO2
+
C11H14NO+
ꢁ5.68
ꢁ3.45
0.96
ꢁ10.08
ꢁ21.90
1.92
C7H7
2.20
+
+
DP-1
C11H14ClNO+
1.89
DP-7
C25H24ClN2O3
C25H22ClN2O2
C
10H11ClN+
0.56
C9H7ClN+
C8H7ClN+
C10H10N+
C9H8N+
ꢁ2.44
+
C
15H12NO2
C7H5O+
8.55
9.02
1.54
8.54
0.60
3.83
1.67
4.48
ꢁ1.89
ꢁ0.29
8.82
DP-8
DP-9
C18H17ClN2O+
C10H9ClN+
C
1.12
2.82
6.25
C8H7N+
+
17H18NO3
+
+
+
DP-2
DP-3
C18H20ClN2O2
C10H10NO3
C18H18ClN2O+
C8H10NO+
ꢁ1.47
ꢁ2.46
2.25
ꢁ5.55
ꢁ4.27
ꢁ6.94
ꢁ8.46
C
10H11ClN+
+
DP-10 C26H24ClN2O3
C8H8NO+
+
C
11H9ClNO2
C7H8N+
DP-11 C10H11ClN+
C9H7ClN+
C19H22ClN2O2
+
10H10N+
C12H13ClNO2
C
C9H8N+
Fig. 4 ESI/MS/MS spectra of (a) DP-6 (m/z 445) at 10 eV, (b) DP-7 (m/z 435) at 15 eV and (c) DP-8 (m/z 313) at 10 eV.
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characteristic fragments for DP-4 include m/z 226 (loss of CO2) spectrum shows characteristic product ions with m/z 427 (loss
and m/z 178 (loss of C7H8). A probable mechanism for the of H2O), m/z 202 (loss of C15H15NO from m/z 427), and m/z 176
formation of DP-4 under hydrolytic conditions may involve (loss of CO from m/z 202) consistent with the structure N-(4-(5-
hydrolytic cleavage of the carbonyl attached to the benzazepine hydroxy-7-methoxy-2,3,4,5-tetrahydro-1H-benzo[b]azepine-1-
moiety of TVT (Scheme 4(a)).
carbonyl)-3-methylphenyl)-2-methylbenzamide. The probable
DP-5. The mass difference between the drug (m/z 449) and mechanism of formation of DP-6 under oxidative degradation is
DP-5 (m/z 431, Fig. 3c) is 18 Da which suggests that it is formed shown in Scheme 4(b).
by the loss of water molecule from the drug during base
DP-7. The ESI-MS/MS spectrum of the [M + H]+ ion (m/z 435)
hydrolysis. The elemental compositions of the [M + H]+ of DP-5 of DP-7 is given in Fig. 4b. The spectrum shows structure
and its product ions (Scheme 3(c)) have been conrmed by indicative product ions which are explained in Scheme 3(b) and
accurate mass measurements (Table 2). All these data are highly listed in Table 2. For example, m/z 417 (loss of H2O) and m/z 105
compatible with the proposed structure N-(4-(7-chloro- (benzylidyne oxonium ion) indicated loss of methylene from
2,3-dihydro-1H-benzo[b]azepine-1-carbonyl)-3-methylphenyl)- TVT (Scheme 4(b)) to form DP-7.
2-methylbenzamide. As shown in the Scheme 4(a), DP-5 may be
formed by base catalyzed elimination of water from TVT.
DP-8. The degradant DP-8 at m/z 313 ([M + H]+) was eluted at
6.24 min. Its MS/MS spectrum (Fig. 4c) displays product ions at
DP-6. Fig. 4a shows the ESI-MS/MS spectrum of the [M + H]+ m/z 206 (((7-chloro-2,3-dihydro-1H-benzo[b]azepin-1-yl)-methyl-
ion (m/z 445) of DP-6 (Rt ¼ 5.52 min) with an elemental idyne)-oxonium), m/z 180 (7-chloro-2,3-dihydro-1H-benzo[b]-
composition of C27H29N2O4. The absence of the chlorine azepine), m/z 134 ((4-amino-2-methylbenzylidene)oxonium) and
isotope pattern in the spectrum conrmed that the lone chlo- m/z 107 (loss of CO from m/z 134) (Scheme 3(b)). The absence of
rine atom was absent in DP-6. As explained in Scheme 3(c), the the product ion at m/z 119 and presence of m/z 134 suggested
Fig. 5 ESI/MS/MS spectra of (a) DP-9 (m/z 284) at 15 eV, (b) DP-10 (m/z 447) at 15 eV and (c) DP-11 (m/z 180) at 15 eV.
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that the probable structure of DP-8 would be (4-amino-2- recovery of TVT in the presence of degradation products ranged
methylphenyl)(7-chloro-2,3-dihydro-1H-benzo[b]azepin-1-yl)- from 99.89 to 100.07%. The intra- and inter-day precisions were
methanone.
determined at three different concentrations, 50, 100 and
DP-9. The ESI-MS/MS spectrum of the [M + H]+ ion (m/z 284) 150 mg mlꢁ1, on the same day (n ¼ 3) and consecutive days (n ¼
of DP-9 (Rt ¼ 6.93 min) with elemental composition of 3), respectively. Table S2† shows that the %RSD values for intra
C
17H18NO3 is given in Fig. 5a. The characteristic fragments of and inter-day precision were <0.41% and 0.56% respectively,
DP-9 (Scheme 3(d)) include m/z 192 (loss of C7H8), m/z 252 (loss indicating that the method was sufficiently precise. The
of OCH3) and m/z 164 (loss of CO from m/z 192). These data robustness of the proposed method was determined by
indicated the structure to be methyl-2-methyl-4-(2-methyl- purposely changing the ow rate (0.25–0.35 ml minꢁ1), column
benzamido)benzoate. DP-9 may be formed by an esterication temperature (30 ꢃ 5 ꢂC) and the % of formic acid in the mobile
of DP-3 due to the presence of methanol under acid hydrolysis phase (0.1 ꢃ 0.02%) at three different concentrations (50, 100,
conditions (Scheme 4(a)).
150 mg mlꢁ1). Each sample was injected in triplicate (n ¼ 3), and
DP-10. DP-10 was formed under oxidative degradation the peak areas obtained were used to calculate means and %
conditions through the oxidation of alcohol to ketone. The ESI- RSD values. The %RSD was <1%. No signicant changes in
MS/MS spectrum (Fig. 5b) of the [M + H]+ ion of DP-10 (m/z 447, assay value were observed by changing these chromatographic
C
26H24NClN2O3, Rt ¼ 7.40 min) displays a product ion at m/z 222 conditions, which conrms the robustness of the method. The
(((7-chloro-5-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-1-yl)- proposed UPLC method was applied to the assay of tolvaptan in
methylidyne)-oxonium ion) (Scheme 3(c)). The presence of TOLVAT tablets and results indicated that the amount of tol-
fragments of m/z 252 and m/z 119 in parallel to those of the drug vaptan was 99.08 ꢃ 0.84 (mean ꢃ S.D.) in the tablets, which
indicated that oxidation occurred at the N-4-(7-chloro-5- corresponds to the requirement of 90–110% according to the
hydroxy-2,3,4,5-tetrahydro-1H-benzo[b]azepine) part of the label claim.
drug. The mechanism of formation of DP-10 is depicted in
Scheme 4(b). Accordingly, the structure of DP-10 may be N-(4-(7-
chloro-5-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepine-1-carbonyl)-
4. Conclusion
3-methylphenyl)-2-methylbenzamide.
The degradation behavior of tolvaptan under various stress
DP-11. The ESI-MS/MS spectrum (Fig. 5c) of the [M + H]+ ion conditions was studied. The drug was found to degrade exten-
of DP-11 (m/z 180, Rt ¼ 7.45 min) displays product ions that are sively to form 4 degradation products under acid and base
compatible with the structure 7-chloro-2,3-dihydro-1H-benzo[b]- hydrolysis and showed only 2 minor degradation products
azepine (Scheme 3(a)). The elemental compositions of DP-11 under oxidative degradation. When methanol was used as a co-
and its product ions have been conrmed by accurate mass solvent in stress studies, 4 degradation products were formed
measurements (Table 2). A probable mechanism for the that were absent when using acetonitrile as a co-solvent. This
formation of DP-11 under acid hydrolysis conditions may suggests that acetonitrile is a preferred co-solvent for stress
involve hydrolytic cleavage of the amide functionality in DP-8 degradation studies. All the degradation products were char-
(Scheme 4(a)).
acterized using ESI/MS/MS in combination with accurate mass
measurements of the product ions and precursors. The degra-
dation pathway of tolvaptan was established.
3.5. Method validation
The stability indicating assay method was validated for speci-
city, linearity, precision (inter-day, intra-day and intermediate
5. Conflicts of interest
precision) and accuracy according to ICH guideline Q2 (R1). The The authors have no conicts of interest in this paper.
specicity of the method was established by determining the
peak purity for TVT and the DPs in a mixture of stressed
samples using a photodiode array (PDA) detector and evaluation
Acknowledgements
of the resolution factor. Peak purity was also demonstrated by The authors are thankful to the Department of Pharmaceuti-
subjecting all the degradation samples to LC-MS. The PDA and cals, Ministry of Chemicals and Fertilizers, Govt. of India, for
mass detector showed an excellent purity across all peaks, providing the funds for research at NIPER, Hyderabad.
which unambiguously proves the specicity of the method. To
establish linearity and range, a stock solution containing 1
References
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Table S2 (see ESI†) shows accuracy data at three different
concentrations in triplicate analysis. The recoveries of the
added drug were obtained from the difference between the peak
areas of the fortied and unfortied degraded samples. The
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